Veterinary vaccines

ABSTRACT

The invention relates to vaccines which are suitable for the prevention of clostridial diseases of sheep (and lambs), providing an effective immunity for up to a year or more following a single injection or dose.

[0001] The present invention relates to novel vaccine compositions forparenteral administration, methods for their use and to processes fortheir preparation.

[0002] Bacterial and viral diseases of sheep, such as Clostridialdiseases, cause considerable economic damage in the agricultureindustry. Vaccination is therefore a very important means of controllingthese diseases

[0003] Many currently available vaccines are comprised of killedantigens, whether inactivated bacterial cells, viral particles orcellular components, absorbed onto alkali earth metal salts (ie.aluminium phosphate or aluminium hydroxide) or as water in oilemulsions. For these vaccines it is recommended that naive animals (ie.animals which have not been previously vaccinated) are treated in a twostage dose regime consisting of an initial dose and a second boosterdose several weeks later. The action of the booster dose raises theantibody titre to a level that may sustain protection from a diseasecausing challenge organism for an extended period. Animals undergoingthis vaccination program are usually mustered each year forrevaccination. Clearly such initial two-dose administration is timeconsuming and expensive and is therefore undesirable.

[0004] The aluminium based vaccines have been found to have relativelyshort duration of protection while water-in-oil emulsion vaccines have alonger duration of protection but have been found to be unsuitable foruse because they cause unacceptable lesions at the injection sites ofthe animals (‘Experimental Clostridial Oil Emulsion Vaccines’ Thomson RO. and Batty I., Bull. Off. int Epiz. 1967 67 (11-12)1569-1581; ‘TheImmunogenicity of a multicomponent Clostridial Oil Emulsion Vaccine insheep’ Thomson et al The Veterinary Record, Jul. 26, 1969). In 1976Jansen et. al. reported the immune response of Cl. botulinum C and Dtoxoids in a water-in-oil emulsion vaccine and noted that the two-stagealuminium based vaccine was not boosted by the second dose to the sameextent as the water in oil compositions (Jansen, B C, Knoetze, P C &Visser, F; Onderstepoort J Vet Res, 43(4) 165-174 (1976)). However, thewater in oil compositions gave an undesirable granulomatous swellingresulting from subcutaneous injection of the vaccine in a largepercentage of animals which is a severe disadvantage for the vaccine'scommercial use.

[0005] WO 91/00106 discloses multi-phase emulsions suitable foradministering active substances or antigens by injection of the water inoil in water type. These emulsions are produced from pharmaceuticallyacceptable emulsifiers which when dissolved in an injectable oil, form ahomogeneous clear phase and have inversion points approaching thetemperature of human or animal bodies. The oils contained in theemulsions include mineral, vegetable or animal oils, and synthetichydrocarbons. It was observed that these vaccines were well tolerated inpigs and did not cause any local reactions, abscesses or necroses.However, no data were provided regarding the level and duration of theimmune responses.

[0006] The applicants provide vaccines which are suitable for theprevention of clostridial diseases of sheep and in particular lambs,providing an effective immunity for up to a year or more following asingle injection or dose. Therefore, the present invention addresses theproblems associated with known vaccines, providing a level of effectiveimmune response in sheep for the period of approximately one year ormore following a single injection or dose of vaccine, The presentinvention also provides vaccines which provide an effective immunityagainst each of a number of diseases for up to a year or more followinga single injection or dose of a multivalent vaccine. The selection of anadjuvant which enhances the antigenic response to clostridial antigensin sheep is thus a problem addressed by the invention. The selection ofan adjuvant which enhances the antigenic response to each of a number ofmicro-organisms in sheep is a particular problem addressed by theinvention.

[0007] Thus according to the present invention there is provided a sheepvaccine composition comprising:

[0008] a) an oily adjuvant acceptable for veterinary purposescomprising:

[0009] i) a white mineral oil having a molecular weight of about 250 to300 and

[0010] ii) a mannitol oleate emulsifier and

[0011] b) an aqueous phase comprising one or more clostridial antigens.

[0012] When used herein the term “sheep” refers to lambs as well asdeveloping and mature sheep. The vaccines of the invention areparticularly useful in the vaccination of lambs.

[0013] The vaccine composition is an injectable emulsion of the water inoil type and preferably has a viscosity of about 200 mPas or less, morepreferably about 100 mPas to about 150 mPas. The white mineral oil ispreferably between about 50% and about 70% by weight of the emulsionmore preferably between about 53% and about 63% by weight of theemulsion. The mannitol oleate emulsifier is preferably between about 2%and about 10% by volume of the emulsion more preferably between about 3%and about 7%.

[0014] The white mineral oil has a molecular weight of about 250 to 300,preferably about 270 to 290, more preferably about 280. The oil ispreferably one which is liquid at 4° C. and has a viscosity lower than100 mPas at 25° C. It preferably has a density at 20° C. of about 815 to840 kg/m³, more preferably about 817 to 837 kg/m³. The dynamic viscosityof the oil at 25° C. is preferably about 5 to 15 mPas, more preferablyabout 6 to 13 mPas. The oil preferably has a kinematic viscosity at 40°C. of about 5 to 10 mm²s, more preferably about 7.5 mm²/s. Preferredembodiments of the invention include the commercially available oilMarcol 52 which is supplied by ESSO.

[0015] The mannitol oleate emulsifier is preferably an anhydromannitolether octadecanoate. Preferred emulsifiers have a viscosity at 25° C. ofabout 300 to 400 cP, more preferably about 340 to about 360 cP,particularly preferred embodiments are those in which the emulsifier hasa viscosity of about 350 cP. The emulsifier preferably has a specificgravity at 20° C. of about 0.8 to 1.0, more preferably of about 0.95 toabout 0.99, particularly suitable are those with a specific gravity at20° C. of about 0.97. Particularly preferred emulsifiers are those witha refractive index at 25° C. of about 1.4 to 1.5, more preferably ofabout 1.47 to 1.48, particularly those with a refractive index at 25° C.of about 1.4748 to 1.4758. Particularly preferred oils are thecommercially available ones Montanide 80, Montanide 103 and Montanide888 supplied by SEPPIC SA, 75 Quai D-Orsay, 75007 Paris. Montanide 103and Montanide 888 being more preferred and Montanide 888 being mostpreferred.

[0016] It will be apparent to a person of skill in the art that theproportion of oily adjuvant to aqueous phase included in the emulsioncan be adjusted to optimise vaccines including particular antigens andfor use in particular animals. It can also be modified to optimisevaccines for administration at a particular site.

[0017] The site of administration may also affect the efficacy and/orthe site reactions caused by the vaccines. It will be apparent to aperson of skill in the art that the site of administration can beselected so as to optimise the effects of vaccines including particularantigens and for use in particular animals. The vaccines exemplifiedherein were found to be efficacious regardless of site ofadministration, however site reactions from the exemplified vaccineswere more numerous when they were administered at the brisket.

[0018] Clostridial antigens suitable for use in the compositions of thepresent invention include Clostridium perfringens type A, B, C and D,Clostridium septicum, Clostridium tetani, Clostridium chauvoei,Clostridium novyi type B, Clostridium sordelli, Clostridiumhaemolyticum, Clostridium chauvoei and Clostridium botulinum C and D.Suitable antigens include those which are useful in the treatment ofdiseases such as Lamb dysentery, Pulpy Kidney disease (enterotosemia),Malignant Oedema (blood poisoning), Tetanus, Blackleg disease and Blackdisease.

[0019] Antigens suitable for use in the present invention are any whichprovide a suitable immune response, eg. toxoids or anacultures. Suitableantigens include Clostridium perfringens A, B, C and D toxoids;Clostridium novyi B toxoid; Clostridium chauvoei anaculture; Clostridiumsepticum toxoid, Clostridium tetani toxoid and Clostridium sordellitoxoid.

[0020] The vaccine is preferably a multi-valent vaccine, ie. a vaccineproviding protection against a number of different clostridial diseasesby incorporating a number of different clostridial antigens eg. thevaccine may contain any number of antigens selected from the listprovided above. It is particularly useful to provide a multivalentvaccine, ie. one which provide adequate immune response to a number ofpathogens to increase the protection provided by the vaccine. It isparticularly difficult to provide multivalent vaccines because it isnecessary to provide a vaccine which induces an adequate antigenicresponse to all the micro-organisms of interest. Thus the thresholdantibody responses are described in compendial standards (eg. AustralianTherapeutic Goods order No. 30; British Pharmacopoeia; EuropeanPharmacopoeia and United States Code of Federal regulation). Wherecompendial standards do not exist (eg. for Corynebacteriumpseudotuberculosis) recognised thresholds based on protection fromchallenge are accepted.

[0021] Preferred embodiments of the invention are vaccines comprising atleast two types of clostridial antigen, each one being active againstany one of the following: Clostridium perfringens; Clostridium novyi;Clostridium chauvoei; Clostridium septicum and Clostridium tetani.Particularly preferred embodiments being vaccines comprising an antigento all five diseases listed.

[0022] Particular embodiments are vaccines comprising at least two ofthe following types of clostridial antigen: Clostridium perfringens Dtoxoid; Clostridium novyi B toxoid; Clostridium chauvoei anaculture;Clostridium septicum toxoid and Clostridium tetani toxoid. Aparticularly preferred embodiment being a vaccine comprising all fiveantigens listed.

[0023] The vaccine may also comprise antigens against other diseases eg.Pasteurella antigens such as Pasteurella maltocida and Pasteurellahaemolyticum; Corynebacterium antigens such as Corynebacteriumpseudotuberculosis, Corynebacterium renale, Corynebacterium cystitis andCorynebacterium pilosum; and Haemophilus antigens such as Haemophilussomnus and Haemophilus pleuropneumoniae; Mycoplasma antigens such asMycoplasma agalactiae and Mycoplasma ovipneumoniae.

[0024] Further preferred embodiments of the invention are those whichcomprise Corynebacterium antigens such as Corynebacteriumpseudotuberculosis, Corynebacterium renale, Corynebacterium cystitis andCorynebacterium pilosum.

[0025] A particularly preferred embodiment of the invention comprisesantigens of Clostridium perfringens D toxoid; Clostridium novyi Btoxoid; Clostridium chauvoei anaculture; Clostridium septicum toxoid,Clostridium tetani toxoid and Corynebacterium pseudotuberculosis.

[0026] The invention particularly relates to vaccines comprising one ormore Clostridial antigens in combination with one or morenon-Clostridial antigens.

[0027] Co-adjuvants may optionally be included in the vaccines of thepresent invention, The antigens may be in the form of toxoids or cellantigens but if cell antigens are used a co-adjuvant may be required.Such co-adjuvants may suitably include a saponin (eg. quil A) orcytokines such as Interleukin-1, 2, and 4 or muramyl dipeptide. Furtheremulsifiers such as dioctyl decyl ammonium bromide (DDA) may also beincluded in the vaccines if desired, The vaccine composition of thepresent invention may contain one or more antigens and one or moreemulsifiers and/or one or more co-adjuvants.

[0028] Supplements such as selenium which is important for growth andreproductive processes may also be included in the vaccine.

[0029] Vaccines according to the present invention may be prepared bydissolving antigens in a suitable aqueous medium such as normal saline,stirring the resultant mixture and adding it to a suitable oil phase,The mixture is then stirred (eg. at 200 to 600 rpm) and/or homogenised(eg. at 500 to 4500 psi) to the desired viscosity (<200 mPas) andconductivity <0.5 millisiemens at 20° C. Preservatives such asthiomersal may optionally be included in the aqueous mixture prior toadding the antigens. This process is preferably carried out at about 20°C. to about 25° C.

[0030] Surprisingly it has been found that the vaccines of the inventioncan provide a sustained and elevated immune response when administeredto the target animals, sheep, in a single dose. They are preferablycapable of inducing a response which can be measured, eg., by ELISA orSN neutralisation titres, for a period of at least 12 months. Thevaccine compositions of the present invention are stable and may bestored for several months or even years without loss of antigenicpotency. The vaccines are capable of overcoming maternal antibody.

[0031] The present invention will now be exemplified with reference tothe following Examples by way of illustration only.

EXAMPLE 1

[0032] Preparation and Efficacy of a Single Dose Clostridial plusCorynebacterium pseudotuberculosis 6-in-1 Vaccine for Use in Sheep

[0033] Vaccine compositions were prepared according to Table I below.The compositions were initially prepared by mixing Clostridial andCorynebacterium pseudotuberculosis antigens with normal saline andthiomersal at room temperature to prepare the aqueous phase of thevaccines. The pH of the aqueous phase being between pH7 and pH7.5.

[0034] For composition 1 the aqueous phase was added to a mixture ofMarcol 52 and Montanide 888 (ratio 10.7 to 1 pre-equilibrated to roomtemperature). This mixture was then homogenised to create a water-in-oilemulsion (viscosity <2 mPas). The dose volume of Composition 1 was 1 mL.

[0035] Composition 2 was prepared in a similar manner except that analuminium based adjuvant, Tasgel was added instead of the Marcol52/Montanide 888 mixture. The aqueous phase/Tasgel mixture was stirredat 100 to 600 rpm at room temperature for 15 minutes and the pH adjustedwith HCl to pH 6.5±0.3. The dose volume of Composition 2 was 2 mL. TABLEI Vaccine Compositions Composition (amount % v/v) Component 1 2 Cl.perfringens D toxoid 5.2 2.60 Cl. novyi B toxoid 5.94 2.97 Cl. chauvoeianaculture 4.4 2.2 Cl. septicum toxoid 1.4 0.7 Cl. tetani toxoid 9 4.5C. pseudotuberculosis toxoid 0.84 0.42 MilliQ 13.9 55.6 Thiomersal 1Marcol 52/ISA 888 58.5 Tasgel 30

[0036] The efficacy of the vaccines was tested using a group of thirtyapproximately four year old pregnant ewes (first cross Border-Leicester)identified by ear tags. The ewes were vaccinated subcutaneously with thevaccines described above approximately two to three weeks prior to theonset of lambing. Twenty lambs born to the previously vaccinated eweswere vaccinated by subcutaneous injection of the vaccine compositions.The vaccine regimes are shown below in Table II: TABLE II Vaccineregimes for ewes and lambs Lambs or No. of Ewes Animals Dose GroupVaccine/adjuvant vaccinated vaccinated vol. (mL) 1 6 in 1/Tasgel Ewes 102 2 6 in I/M52-M888 Ewes 20 4 Non-vaccinate (control) Lambs  4 — 5 6 in1/Tasgel Lambs  5 2 6 Non-vaccinate(control) Lambs 11 — 7 6 in1/M52-M888 Lambs 15 1

[0037] Lambs were at least eight weeks old when vaccinated. Only Group 5received a second vaccination at Week 4, these animals were vaccinatedwith 6 in 1 vaccine comprising Tasgel as adjuvant.

[0038] Serum samples were collected from blood centrifuged for 15minutes at 300 rpm and at room temperature. The following assays wereperformed on serum samples:

[0039]Cl. perfringens D ELISA was performed on individual serum samplesusing purified rabbit anti-Cl. perfringens epsilon toxin diluted incarbonate buffer pH9.6 in a 96 well microtitre plate. This was incubatedat 37° C. for 2 h before purified Cl. perfringens toxin diluted inphosphate buffered saline/Tween 20 (0.1% w/v) was added and the plateincubated at 37° C. for 1 h. Dilutions of sheep sera (and positive andnegative sera) were added to the wells and the plate incubated for 1 hat 37° C. The plates were then washed with PBS/Tween and a dilution ofrabbit anti-sheep IgG horseradish peroxidase conjugate (Biorad) inPBS/Tween added. The plate was incubated at 37° C. for a further hourand then washed with PBS/Tween. Activated substrate(2,2-azino-di-3-ethylbenzthiazolinosulfonate) dissolved in citratephosphate buffer pH4.6 at 1 mg/mL and activated by addition of 0./3%hydrogen peroxide was added to the plate. Absorbance at 405 nm was readafter 30 to 60 minutes using a Titertek Multiscan reader.

[0040] The procedure was repeated using purified Cl. tetani toxoid inthe Cl. tetani ELISA on individual serum samples.

[0041] Mice serum neutralisation titres of pooled group sera for Cl.novyi, Cl. tetani, Cl. septicum and Cl. perfringens D were carried outaccording to the Therapeutic Goods Order No. 30 (Australian GovernmentPublishing Services, 1987).

[0042]Corynebacterium pseudotuberculosis (C. pseudotuberculosis) serumneutralisation titres were determined for individuals and pooled serasamples based on observations by Muckel & Giles, Am. J. Vet. Res. 44,1149-1153, 1983 in order to measure a response to the Corynebacteriumpseudotuberculosis. antigens.

[0043] The pregnant ewes were monitored on Day 0, Day 1 and Day 14 (Week2) following vaccination. No ewes appeared distressed. Rectaltemperatures were recorded on those days and a summary is shown in TableIII. TABLE III Rectal temperatures of pregnant ewes prior to andfollowing vaccination Mean rectal temperature (° C.) Day Groupvaccine/adjuvant 0 1 14 1 6 in 1/Tasgel 39.7 ± 0.3 40.0 ± 0.5 39.6 ± 0.32 6 in 1/M52-M888 39.7 ± 0.2 40.2 ± 0.5 39.5 ± 0.3

[0044] Site reactions were recorded for the lambs and are shown below inTable IV. They were found to be negligible for each type of vaccination.TABLE IV Mean site reactions (cm³) following vaccination of lambs GroupWeek 4 Week 8 Week 12 Week 20 Week 26 Week 30 4 Non vaccinates 0 (0/3) 50 (0/5) 0 (0/5) 0 (0/5) 0 (0/5) 0 (0/5) 0 (0/5) 6 Non vaccinates 0 (0/6)7 1.8(1/13) 0.5(1/15) 0.6(1/14) 0(0/15) 0(0/5) 0(0/8)

[0045] The figures presented in brackets represent the number of lambswith site reactions/number of lambs per group.

[0046] Selected groups were revaccinated at Week 26 and no sitereactions were reported at Week 30. The weights of lambs were recordedand are shown below in Table V. No significant difference in weights orweight gain based on 95% confidence interval of the mean for each groupat each time point and between non-vaccinates and vaccinates was found(p >0.05). TABLE V Weights of lambs (kg) Average weight at week Averageweight gain Group Marking 4 8 12 over 12 weeks 4 21.6 ± 4.0 31.6 ± 4.235.0 ± 5.0 42.3 ± 5.5 19.8 ± 2.1 5 22.1 ± 2.5 31.1 ± 3.4 33.5 ± 3.7 38.2± 3.9 16.1 ± 1.6 6 19.6 ± 5.2 27.9 ± 5.3 30.8 ± 5.9 35.1 ± 6.2 15.5 ±2.5 7 21.3 ± 3.5 30.8 ± 3.8 32.6 ± 3.9 37.4 ± 3.7 16.1 ± 3.2

[0047]Cl. perfringens D ELISA Titres

[0048]Cl. perfringens D ELISA titres were determined for both ewes andlambs to evaluate vaccine efficacy. Tables VI and VII record Cl.perfringens D ELISA titres for pregnant ewes and lambs (respectively).Groups 4 and 5 were given a booster vaccination of 6 in 1 vaccine withTasgel adjuvant at Week 26. Tables VIII and IX show SN's for pregnantewes and lambs (respectively) TABLE VI Cl. perfringens D ELISA (U/mL) ofewes Week Group adjuvant Week 0 Week 17 37 1 6 in 1 Tasgel 1 × 2 mL dose1.4 4.3 8.7 6 6 in 1 M52-M8881 × 1 mL dose 2.6 24.4 23.2

[0049] The results displayed are the GMT of individual titres for thatgroup. TABLE VII Cl. perfrigens D ELISA Titres in lambs Mean ELiSATitres (U/mL) Group 0   4   8   12   20   26 ,,˜30 4 1.1 <1 <1 <1 <1 <15 2.0 <1 <1 <1 <1 <1    1.3 6 3.2 <1 <1 <1 <1 <1 7 2.2   2.1   3.4   3.9  4.4   2.8    3.5

[0050] TABLE VIII SN Titres in pregnant ewes Group adjuvant 17 weeksafter vaccination 1 6 in 1 Tasgel 1 × 2 mL Cl. perf D  5.5-11 dose Cl.tet   8-10 Cl. novyi B   6-8 Cl. sept  <2 2 6 in 1 M52-M888 1 × 1 mL Cl.perf D 13.2-16.5 dose Cl. tet >12 Cl. novyi B   3-5 Cl. sept >10

[0051] TABLE IX SN Results for lambs Group Vaccine Week 0 Week 8 5 6 in1 Tasgel Cl. perf D   1.7-2.5 Cl. perf D <3.65 2 × 2 mL dose Cl. tet  2.5-3.8 Cl. tet <2 Cl. novyi B   2.3-3.5 Cl. novyi B   1.5-2.3 Cl.sept <2 Cl. sept <2 C. pseudot   2.1 C. pseudot   0.2 7 6 in 1 M52- Cl.perf D <1.1 Cl. perf D <3.6 M888 1 × 1 mL Cl. tet   1.7-2.5 Cl. tet  2.5-3.8 dose Cl. novyi B   1.5-2.3 Cl. novyi B   2.3-3.5 Cl. sept <2Cl. sept <2 C. pseudot   1.2 C. pseudot   0.1 Group Vaccine Week 26 Week30 5 6 in 1 Tasgel Cl. perf D <1.1 Cl. perf D <4 2 × 2 mL dose Cl. tet<1.1 Cl. tet >4.1 Cl. novyi B <1.0 Cl. novyi B   7.1-10.7 Cl. sept <2.0Cl. sept <2.0 C. pseudot   0.8 7 6 in 1M52- Cl. perf D   1.1-1.7 M888 1× 1 mL Cl. tet <1.1 dose Cl. novyi B <1.0 Cl. sept <2.0 C. pseudot 1.1

[0052] Vaccination of lambs in Group 7 with the single dose M52-M888adjuvanted vaccine (the same vaccines as their mothers were vaccinatedwith) indicated that maternal antibody could be overcome by providing aresponse to vaccination, when compared to the standard (Group 5).Comparing non-vaccinates with vaccinates (Groups 4 and 5 or groups 6 and7) maternal antibody appeared to decline to a level below thesensitivity of the ELISA by Week 4 following marking. The 95% confidenceintervals of the mean for each group indicate that from Week 8 onwardsonly Group 7 has a statistically significantly higher titre than allother groups (p <0.05). Thus, based on these ELISA results and similaranalysis for other Clostridial and bacterial components, the capacity ofa single dose of the M52-M888 adjuvanted vaccine to generate responsesis at least as good as the two dose vaccine. Furthermore the M52-M888adjuvanted vaccine was administered in a single dose.

EXAMPLE 2

[0053] Preparation and Efficacy of a Single Dose Clostridial 5-in-1Vaccine for Use in Sheep

[0054] Vaccine compositions used in this example were prepared in asimilar manner to the compositions described above in Example 1 (seeTable I) except that the C. pseudotuberculosis antigen was omitted.Selenium (1 mg per dose) was added to the aqueous phase prior to mixingwith either Marcol 52/Montanide 888 or Tasgel.

[0055] Fine wool Merryville Merino ewes and lambs, identified bynumbered ear tags, were vaccinated subcutaneously in the left side ofthe neck in accordance with the regime set out below in Table X. Theywere vaccinated on the right side of the neck when a second andlorbooster dose was required. All these lambs were derived from Merino ewesthat had been previously vaccinated with 5 in 1 Tasgel vaccine 1 monthprior to lambing. TABLE X Vaccine groups Dose Group VaccineAntigen/Adjuvant No. of Lambs Volume (mL) 1 Non-vaccinates 10 — 2 5 in1/Tasgel 18 2 3 5 in 1 + Se/Tasgel 18 2 4 5 in 1/M52-M888 18 1 5 5 in1Se/M52-M888 18 1

[0056] Lambs were vaccinated when four to eight weeks old. Groups 2 and3 were vaccinated with a second dose of 5 in 1 Tasgel vaccine at week 4.Groups 2, 3, 4 and 5 received a booster vaccination with the respectivevaccinates for the group at week 51.

[0057] Serum samples were collected from blood centrifuged at 3000 rpm,15 min at room temperature. The assays described above in Example 1 wereperformed on the serum samples.

[0058] Site reactions following vaccination were not found in any of theMerino lambs. No adverse affects on the lambs' general well being as aresult of vaccination were observed.

[0059]Cl. perfringens D and Cl. Tetani ELISA Titres

[0060] The response of the lambs to vaccination was assessed bymeasurement of Cl. perfringens D and Cl. tetani antibody responses usingspecific ELISAs. Cl. Perfringens D results are presented below in TableXI and Cl. tetani results are presented in Table XII. TABLE XI Cl.perfringens D ELISA Titre in Lambs (U/mL) Cl. perfringens ELISA Titre(U/mL) Vaccine Antigens/ Vaccination WEEK Group Adjuvant (week) 0 4 8 1219 26 51 55 1 Non-vaccinate 1.6 <1 <1 <1 <1 <1 <1 <1 2 5 in 1 Tasgel 0,4 2.2 <1 <1 2.9 <1 <1 <1 1.4 3 5 in 1 +Se/Tasgel 0, 4 3.6 <1 <1 1 <1 <1<1 2.4 4 5 in 1 M52/M888 0 2.7 5.1 11.5 17 80 13.6 12.6 15.6 5 5 in 1+SeM52/M888 0 <1 1.3 4.8 9.2 20 10 8 16.8

[0061] TABLE XII Cl. tetani ELISA Titre in Lambs (U/mL) Cl. tetani ELISATitre (U/mL) Vaccine Antigens/ Vaccination WEEK Group Adjuvant (week) 04 8 12 19 26 51 55 1 Non-Vaccinate 1.9 <1 <1 <1 <1 <1 <1 <1 2 5 in1/Tasgel 0, 4 <1 <1 <1 <1 <1 <1 <1 :1.9 3 5 in 1 +Se/Tasgel 0, 4 <1 <1<1 <1 <1 <1 <1 1.6 4 5 in 1 +/M52-M888 0 <1 <1 4.5 10.4 2.3 3.4 1.3 10.45 5 in 1 +Se/M52-M888 0 1.4 <1 5.2 5.6 1 2.4 1.5 10.4

[0062] Both Cl. perfringens D and Cl. tetani ELISA titres in iambs weredetected over 51 weeks following a single dose vaccination with theM52-M888 adjuvanted vaccine formulation. In comparison, ELISA titreswere not-detectable following two doses of the 5 in 1 gel vaccine. Thismay be due to the levels of maternal antibody present in the lambs. InGroup 4 where maternal antibody was detected at marking (week 0), theM52-M888 adjuvanted vaccine formulation overcame maternal antibody toproduce a strong response.

[0063] The M52-M888 adjuvanted vaccines showed higher efficacy than the5 in 1 Tasgel adjuvanted vaccine formulations. Furthermore the M52-M888adjuvanted vaccine formulation (Group 4) was used in a single doseregime compared to a two dose regime for the gel formulations (Groups 2and 3). The addition of selenium did not offer any immunologicaladvantage, although it is known to act as an immune stimulant. It isprimarily added as a mineral supplement to these vaccines.

[0064] SN Titres in Lambs

[0065] SN titres for Cl. perfringens D, Cl. tetani, Cl. septicum and Cl.novyi B were determined for selected pooled group sera samples fromGroups 1 to 5. At Week 12, only lambs vaccinated with the 5 in 1M52/M888 adjuvanted vaccine formulation (Group 4) gave detectable SNtitres for the components Cl. perfringens D, Cl. tetani, Cl. septicumand Cl. novyi B. By Week 19 SN titres for Cl. tetani and Cl. novyi Bremained detectable for this formulation. Results are presented below inTable XIII. Pooled group sera from lambs vaccinated with formulationscontaining selenium were only assayed at Week 12 for Cl. perfringens Dand Cl. tetani. Results were similar to that found in lambs vaccinatedwith formulations without selenium. TABLE XIII Serum neutralisationtitres in lambs Vaccine Vaccination Group (Antigen/Adjuvant) (week)Antigen Week 0 Week 4 Week 8 Week 12 1 Non-vaccinates Cl. perfringens D<3.6 <1.4 Cl. tetani <2.0 <2.2 Cl. septicum NA NT NT NT Cl. novyi B <1.5NT 2 5 in 1 Tasgel 0, 4 Cl. perfrigens D <3.6 <3.6 <3.6 <1.4 Cl. tetani<2.0 <2.0 <2.0 <2.2 Cl. septicum <2.2 <2.2 <1.80 <1.8 Cl. novyi B  1.5-2.3 <1.5 2.3-3.5 <1.6 3 5 in 1 + 0, 4 Cl. perfrigens D <1.4 SeTasgel Cl. tetani <2.2 Cl. septicum NT NT NT NT Cl. novyi B NT 4 5 in 10 Cl. perfrigens D <3.6 3.6-4.5 9.0-13.6 4.6-6.9 M52-M888 Cl. tetani<2.0 2.5-3.8 8.6 6.2-9.3 Cl. septicum <2.2 <2.2 2.3-3.4 2.3-3.4 Cl.novyi B <1.5 3.5-5.3 8.0-12.0 5.3-7.9 5 5 in 1 + 0 Cl. perfrigens D 3 SeM52-M888 Cl. tetani 2.8-4.1 Cl. septicum NT NT NT NT Cl. novyi B NTVaccine Vaccination Group (Antigen/Adjuvant) week Antigen Week 19 Week51 Week 55 1 Non-vaccinates Cl. perfrigens D <3.0 <1.1 Cl. tetani <2.2<1.2 Cl. septicum <1.8 <2 NT Cl. novyi B <1.4 <1 2 5 in 1 Tasgel 0, 4Cl. perfrigens D <3.0 1.1 <1.1 Cl. tetani <2.2 <1.2 NA Cl. septicum <1.8<2 <2 Cl. novyi B <1.4 <1 3.5-5.3 3 5 in 1 + 0, 4 Cl. perfrigens D SeTasgel Cl. tetani Cl. septicum NT NT NT Cl. novyi B 4 5 in 1 0 Cl.perfrigens D <3.0 <1.1 >8.8 M52-M888 Cl. tetani 4.1-6.2 <1.2 1.8-2.8 Cl.septicum <1.8 <2 8.6-12.9 Cl. novyi B 2.1-3.2 <1 >8 5 5 in 1 + 0 Cl.perfrigens D Se M52-M888 Cl. tetani Cl. septicum NT NT NT Cl. novyi B

[0066] Rabbit Potency

[0067] The vaccines were assessed in the standard rabbit potency test(Therapeutic Goods Order No. 30 Australian Government Publishing 1987).A summary of results is presented in Table XIV. The vaccine formulationof 5 in 1 M52-M888 adjuvanted vaccine formulation (Group 4) passedpotency in all fractions, following a single dose vaccination. Thisresult was similar to that of the standard gel vaccine following twodoses (Group 2). The vaccines with selenium (Groups 3 and 5) were notassessed in the rabbit potency test. TABLE XIV Initial Rabbit Potency ofVaccines Vaccine Antigens/ SN U/mL Group Adjuvant Cl. tetani Cl.perfringens D Cl. septicum Cl. novyi B 2 5 in 1/Tasgel 2.8-4.1 9-13.55.6-8.4 4.7-7.1 4 5 in 1/M52-M888 9.3-13.9 20.2-30.4 3.8-5.6 >10.7 CODEXPASS 2.5 5 2.5 3.5

[0068] Rabbit Potency Shelf Life

[0069] The 5 in 1 M52-M888 adjuvanted vaccine formulation (Group 4) wasassessed for stability at 2, 14 and 22 months post manufacture in therabbit potency test. Results are presented below in Table XV. Theypassed potency in all fractions. TABLE XV Rabbit Potency Shelf LifeVaccine Antigens/ Shelf Life at SN U/mL Adjuvant Test (Months) Cl.tetani Cl. perfringens D Cl. septicum Cl. novyi B 5 in 1 Tasgel 2  2.8-4.1   9-13.5   5.6-8.4   9.7-7.1 14   2.5-3.8   6-9   7.6-11.4  3.5-5.3 5 in 1 M52-M888 2   9.3-13.9   20.2-30.3   3.8-5.6 >10.7 14  11.2   18.7-20.1 >11.6 >12 22 >12.9 >28   9.514.2 >12 CODEX PASS 2.5  5   2.5   3.5

[0070] Guinea Pig Cl. chauvoei Challenge

[0071] The Cl. chauvoei guinea pig challenge test is a regulatoryrequirement (Therapeutic Goods Act, 1966, Therapeutic Goods Order No.30). Vaccines with a Cl. chauvoei fraction must pass this test in orderto be released for use. A pass in the challenge test is at presentdefined as: “10 out of 10 guinea pigs vaccinated with two doses (4 weeksapart) of an aluminium hydroxide adjuvanted clostridial vaccine,surviving a Cl. chauvoei challenge at 2 weeks post second vaccinationfor 5 days post challenge”. This result is compared with non-vaccinatedcontrols which must succumb to challenge within 3 days. The vaccineswere assessed in the Cl. chauvoei guinea-pig challenge test and theresults of the challenge tests are shown below in Table XVI. TABLE XVIGuinea Pig Cl. chauvoei Challenge Survivors/ Pass/Fail GroupVaccine/Adjuvant No. Challenged (Challenge) 4 5 in 1 M52-M888  5/5,10/10 Pass CODEX PASS 10/10

[0072] The results discussed above indicate that single dose vaccine ismore efficacious and provides a sustained immune response to theclostridial antigens as determined by ELISA and SN tests.

[0073] The single dose vaccine is safe, with negligible site reactionsand passed the rabbit potency test in all fractions (Cl. perfringens D,Cl. tetani, Cl. septicum, Cl. novyi B and Cl. chauvoei). and the guineapig Cl chauvoei challenge test.

1. A sheep vaccine composition comprising: a) an oily adjuvant acceptable for veterinary purposes comprising: i) a white mineral oil having a molecular weight of about 250 to 300 and ii) a mannitol oleate emulsifier and b) an aqueous phase comprising one or more clostridial antigens.
 2. A vaccine composition as claimed in claim 1 wherein the vaccine has a viscosity of about 200 mPas or less.
 3. A vaccine composition as claimed in claim 1 or claim 2 wherein the white mineral oil is between about 50% and about 70% by weight of the emulsion.
 4. A vaccine composition as claimed in any one of claims 1 to 3 wherein the mannitol oleate emulsifier is between about 2% and about 10% by volume of the emulsion.
 5. A vaccine composition as claimed in any one of claims 1 to 4 wherein the mannitol oleate emulsifier is an anhydromannitol ether octadecanoate.
 6. A vaccine composition as claimed in claim 5 wherein the vaccine comprises one or more of the following Clostridial antigens: Clostridium perfringens type A, B, C and D, Clostridium septicum, Clostridium tetani, Clostridium chauvoei, Clostridium novyi type B, Clostridium sordelli, Clostridium haemolyticum and Clostridium chauvoei.
 7. A vaccine composition as claimed in any one of claims 1 to 6 wherein the vaccine comprises antigens useful in the treatment of one or more of the following: Lamb dysentery, Pulpy Kidney disease (enterotoxaemia), Malignant Oedema (blood poisoning), Tetanus, Blackleg disease and Black disease.
 8. A vaccine composition as claimed in any one of claims 1 to 7 wherein the vaccine further comprises antigens against a non-clostridial disease.
 9. A vaccine composition as claimed in claim 8 wherein the vaccine comprises Corynebacterium antigens. 